Low Density Lipoprotein Degradation by Secretory Granules of Rat ...

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The secretory granules of rat serosal mast cells are able efficiently to degrade the apolipoprotein B com- ponent of low density lipoproteins (LDL) (Kokkonen,.
Vol. 261, No. 34, Issue of December 5, pp. 16067-16072 1986 Printed in S. A .

THEJOURNAL OF BIOLOGICAL CHEMISTRY

ii.

0 1986 by The American Society of Biological Chemists, Inc.

Low Density Lipoprotein Degradation bySecretory Granules of Rat Mast Cells SEQUENTIALDEGRADATION CARBOXYPEPTIDASE A*

OF APOLIPOPROTEIN B BY GRANULECHYMASE AND

(Received for publication, June 16, 1986)

Jorma 0. Kokkonen, Maire Vartiainen, and Petri T.KovanenS From the Wihuri Research Institute, Kallwlinnnntie4, SF-00140 Helsinki, Finland

The secretory granules of rat serosal mast cells are peptidase A (Woodbury et al., 1981). Both of these enzymes able efficiently to degrade the apolipoprotein B com- have been isolated and studied inpurified form. Chymase, as ponent of low density lipoproteins (LDL) (Kokkonen, an endopeptidase with a-chymotrypsin specificity, is able to J. O., and Kovanen, P. T. (1985) J. Biol. Chern. 260, cleave severalnaturally occurring proteins, e.g. glucagon, neu14756-14763). The granules are known to contain rotensin, human plasma fibronectin, and type IV collagen two neutral proteases with complementary specifici- (Schwartz and Austen, 1984). In contrast, the purified granule ties: a chymotrypsin-like endopeptidase called chy- carboxypeptidase A, with a specificitysimilar to that of bovine mase, and an exopeptidase, the granule carboxypepti- carboxypeptidase A, has been shown to cleave several syndase A. The role of this enzyme pair in the proteolytic thetic dipeptides (Everitt and Neurath, 1980). Because the degradation of LDLwas studied with the aid of specific two enzymes have complementary specificities they could act enzyme inhibitors. Incubation of LDL with intact gran- in concert (Schwartz andAusten, 1984). However, no studies ules (both enzymes active) led to the formation of numerous low molecular weight peptides and the libera- on the interplayof the granule-bound chymase and carboxytion of free amino acids, mostof which (95%) were peptidase A have been reported andconsequently, the biologaromatic (Phe, Tyr, Trp) or branched-chain aliphatic ical actions of this enzyme pair have remained obscure. We recently observed that stimulation of rat serosal mast (Leu, Ile, Val). Selective inhibition of granule carboxcells by the compound 48/80 and the ensuing degranulation ypeptidase A (leaving chymase active) blocked the liberation of free amino acids, but left the formation of result in efficient extracellular proteolysis of LDL’ (Kokkopeptides uninhibited. On the other hand, selective in- nen and Kovanen, 1985). The proteolytic degradationof the hibition of granule chymase (leaving carboxypeptidase apolipoprotein B of LDL was shown to be causedby the A active) totally abolished the proteolytic degradation extruded mast cellgranules. The present studies were deof LDL. The results are consistent with a model ac- signed to test whether thetwo proteolytic enzymes present in cording to which the proteolytic degradation of LDL the granules are involved in theobserved degradation of LDL. by mast cell granules results from coordinated action The results show that the degradationof apolipoprotein B of of the two granule-bound enzymes, whereby the chy- LDL by mast cell granules results from the concerted action mase first cleaves peptides from the apolipoprotein B of the granule chymase andcarboxypeptidase A. of LDL, and thereafter the carboxypeptidase A cleaves amino acids from the peptides formed. EXPERIMENTAL PROCEDURES~ RESULTS

The function of mast cell granules as carriersof mediators of theimmediate-typehypersensitivityreactions is well known. Thus, on antigen challenge, the mast cells are activated and the secretory granules containing the mediators extruded into the extracellular space (Ishizaka and Ishizaka, 1984). After degranulation, thesoluble mediators arereleased from the granules anddiffuse away t o exert their functions in the allergic reactions. In contrast, the major protein component of the granules, consisting of neutral proteases, is not released but remains tightly bound to the heparinproteoglycanmatrix of thegranules, so forming anextracellularly locatedinsoluble heparin-protease complex (Schwartzand Austen, 1984). The protease component of the secretory granules of rat serosal mastcells consists of two proteases,a n endopeptidase called chymase and an exopeptidase called granule carboxy* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ T o whom correspondence should be addressed.

Analysis of the lZ5I-LDL Degradation Products-Upon incubation of lZ5I-LDLwithisolated mast cell granules, the apolipoprotein B component of LDL is rapidly degraded to are molecularweight degradationproducts which can be low readily separated from the lZ5I-LDLby trichloroacetic acid treatment (Kokkonen and Kovanen, 1985). To separate the degradation products from the residual LDL particles by a mild method, gel filtration was used in the presentwork. For this purpose, 100 pg of “‘1-LDL were first incubated with 20 pg of isolated granules at 37 “C for 6 h. After incubation, the ’The abbreviations used are: LDL, low density lipoproteins; HPLC, high performance liquid chromatography; SDS, sodium dodecyl sulfate. Portions of this paper (including “Experimental Procedures” and Fig. 6) are presented in miniprint at theend of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD20814. Request Document No. 86M-2006, cite the authors, and include a check or money order for $1.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

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L D L Degradation by Mast Cell Granules

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granules were sedimented t o remove the proteolytic enzymes and the supernatant was applied to a column of Bio-Gel A5m. As shown in Fig. M, the elutionprofile of the supernatant showed two peaks. Peak I material emerged in the position corresponding to native LDL, and peak I1 material emerged inthepositioncorrespondingtothetotal volume of the column. When the fractions containing peak I and peak I1 material were separately pooled and treated with trichloroacetic acid, it appeared that all of the radioactive material in peak I was acid-precipitable, whereas all of the material in peak I1 was acid-soluble. The amountof radioactivity in peak I1 represented about 37% of the total radioactivity originally present in the incubation system. On the other hand, when lZ5I-LDLwas incubated without mast cell granules, only a minor peak I1 was present (Fig. 1B). T o further characterize the degradation products of lZ5ILDL, thepooled peak I1 material was analyzed on a Sephadex G-10 column (exclusion limit 800). The elutionprofile of peak I1 material disclosed essentially four peaks (Fig. 2 4 ) . Peak a contained material emerging at the void volume, suggesting that it contained a mixture of peptides formed during the course of lZ5I-LDL degradation. The two minor peaks ( b and c) in the Sephadex G-10 eluate, which were also present in the "'I-LDL preparation incubated without granules (Fig. 2B) consisted of free iodine, as shown by three independent methods. First, thin layer chromatography of the material showed that it had the same RF value as commercial NalZ5I; second, the elution profile of commercial NalZ5I showed two major peaks in the positions of peaks b and c; and third, after treatment with H,Oz all of the radioactivity contained in

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FIG. 1. Bio-Gel A-5m analysis of "'I-LDL degraded by mast cell granules. 100 pg of '"I-LDL (50 cpm/ng) were incubated at 37 "C for 6 h in 250 pl of buffer A containing 20 pg of granules ( A ) . In a control experiment no granules were added ( B ) .The reactions were stopped by adding 250 pl of ice-cold buffer A and removing the granules by centrifugation at 12,000 X g for 5 min. The supernatants were applied to a Bio-Gel A-5m column (1X 18 cm) equilibrated with buffer A. The column was eluted with buffer A at a flow rate of 3.4 ml/h a t 4 'C. Fractions of about 280 pl were measured for their lz5I radioactivity. Fractions 43-55 (the shaded area of peak 11) were pooled and further analyzed on a Sephadex G-10 column (Fig. 2). The total volume of the column (V,)and theelution position of native LDL are indicated by arrows.

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FIG. 2. Sephadex G-10 analysis of the labeled degradation products of '"I-LDL. 2 ml of the peak I1 material obtained from the Bio-Gel A-5m analysis (Fig. L 4 , containing 1,780,000 cpm) was applied to a Sephadex G-10 column (A). Panel B shows the corresponding analysis of the peak 11 material represented in Fig. 1B (containing 354,000 cpm). The column (1 X 30 cm) equilibrated with buffer A was eluted with the same buffer at a flow rate of 5 ml/h a t 4 "C. Fractions of about 370 pl were measured for their '*'I radioactivity. The void volume of the column (V,) as well as the elution positions of commercial NalZ5Iand commercial 3-iodo-~-tyrosine are indicated by arrows. The inset shows the results of thin layer chromatography of the peak d material obtained from G-10 column analysis. An aliquot (2800 cpm) of the pooled peak d material (fractions 66-85) was mixed with 30pgof unlabeled 3-iodo-~-tyrosine. Aliquots of this mixture were chromatographed on cellulose sheets with butano1:acetic acid:water (4:l:l) asthe developingsolvent system as described under "Experimental Procedures." The sheets were cut into 1-cm strips, which were counted for their ''I radioactivity. The shaded spot indicates the region containing the unlabeled S-iodo-~tyrosine.

peaks b and c could be extracted with chloroform (Bierman et al., 1974). Most of the lZ5I-labeledmaterial applied to the Sephadex G-10 column was recovered in peak d . When unlabeled 3iodo-L-tyrosine was chromatographed on Sephadex G-10, it emerged at an elution volume identical to that of peak d, suggesting that the material peak in d was monoiodotyrosine. The presenceof monoiodotyrosine in peak d could be verified by thin layer chromatography, which showed that the material ofpeak d had the sameRFvalue as 3-iodo-~-tyrosine (Fig. 2 A , inset). Determination of the Activities of Chymase and CarboxypeptidaseA in Isolated Granules-The demonstration of the presence of the free amino acid lZ5I-tyrosine among the degradation products of lZ5I-LDLsuggested the presence of a functional exopeptidase, namely carboxypeptidase A, in the isolated mast cell granules. To be able to determine theroles of the two proteolytic enzymes of the granules in thedegradation of lZ5I-LDL, conditionswere worked out whereby the activities of either enzyme could be measured selectively. For this purpose, specific low molecular weight substrates and inhibitors of both enzymes were used (Fig. 3). Accordingly, mast cell granules were incubated either with N-benzoyl-Ltyrosine ethyl ester to measure the activityof chymase (Fig. 3A) or with hippuryl-L-phenylalanineto measure the activity

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L D L Degradation by Mast Cell Granules A. No inhibitors

A. Chymase

o,175

0 25

0.150 0.125

.-cE

-0

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am

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'+o-Phenanthroline

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+Trypsin inhibitor

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.-CE

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Granules $g/assay) FIG. 3. Measurement of chymase (A) and carboxypeptidase A ( B ) activity in mast cell granules. Granules (70 pg)were incubated at 37 "C for 3 h in 100 pl of buffer A containing 2 mM ophenanthroline dissolved in 25% (v/v) methanol to give a final concentration of 2.5% methanol (A). In a parallel incubation, 70 pg of granules were incubated in the same buffer containing 2.5% methanol for 3 h after which 750pgof soybean trypsin inhibitor were added (0).In a control incubation, 70 pg of granules were incubated in the same buffer containing 2.5% methanol without inhibitors (0). The chymase and carboxypeptidase A activities of the o-phenanthroline-treated, trypsin inhibitor-treated, or control granules were determined spectrophotometrically as described under "Experimental Procedures."

of carboxypeptidase A (Woodbury et al., 1981) (Fig. 3B). In addition, granules were incubated with either soybean trypsin inhibitor or o-phenanthroline to inhibit the activities of chymase or carboxypeptidase A, respectively. As shown in Fig. 3, both substrateswere hydrolyzed by purified mast cell granules indicating that theycontained active chymase and active carboxypeptidase A. In addition, it could be demonstrated that each enzyme was completely inhibited by its specific inhibitor, the otherenzyme being unaffected. Effect of Inhibition of Granule Chymase and Carboxypeptidase A on Degradation of '25Z-LDL-To assess the respective roles of granule chymase and carboxypeptidase A in the degradation of LDL, lZ5I-LDLwas incubated with purified mast cell granules in the presence of the selective inhibitor of one or the other enzyme. The formation of radiolabeled low molecular weight degradation products was analyzed by sequential fractionationon columns of Bio-Gel A-5m and Sephadex G-10 (Fig. 4). Fractionation on Bio-Gel A-5m showed that selective inhibition of carboxypeptidase A did not affect the formation of the degradation products (Fig. 4B),whereas selective inhibition of granule chymase totally abolished the degradation of lZ51-LDL(Fig. 4C). An identical elution profile with that observed after chymase inhibition was obtained if lZ5I-LDLwas incubated in the absence of granules (Fig. 40). Further analysis of the degradation products on a Sephadex G-10 column (Fig. 5 ) disclosed that the inhibition of granule

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FIG. 4. Effect of o-phenanthroline and trypsin inhibitor on the lZ6I-LDLdegradation by mast cell granules. Granules (30 pg) were preincubated with o-phenanthrolineor soybean trypsin inhibitor or without inhibitors as described in the legend to Fig. 3. The degradation assay was conducted in 250 pl of buffer A containing 100 pg of '=I-LDL (27 cpm/ng) and 20 pg of control (A), o-phenanthroline-treated ( B ) ,or trypsin inhibitor-treated (C) granules. In an additional experiment no granules were added (D). After incubation at 37 "C for 3 h, the reactions were stopped by adding 250 pl of icecold buffer A and removing the granules by centrifugation at 12,000 X g for 5 min. Each supernatant was applied to a Bio-Gel A-5m column (1 X 13 cm) equilibrated with buffer A. The column was eluted with buffer A at a flow rate of 3.4 ml/h at 4 "C. Fractions of about 280 r l were measured for their lz5Iradioactivity. Fractions 2939 (the shaded area of peak 11) were pooled and further analyzed on a Sephadex G-10 column (Fig. 5). The total volume of the column (V,)and theelution position of native LDL are indicated by arrows.

carboxypeptidase A had blocked the production of '251-tyrosine but not that of the lZ51-labeledpeptides (Fig. 5B). If granule chymase was inhibited, the only radioactive material present in the eluate was free radiolabeled iodine (Fig. 5C). An identical elution profile was obtained if lZ51-LDLwas incubated in the absence of granules (Fig. 50). Thus, for the formation of lZ51-labeledpeptides only chymase activity was needed, whereas for the formation of '251-tyrosineboth chymase and carboxypeptidase A activities were necessary. The results are compatible with a model according to which granule chymase and carboxypeptidase A act in sequence, whereby the chymase first cleaves lZ5I-labeledpeptides from the apolipoprotein B of lZ51-LDLand the granule carboxypeptidase A subsequently liberates '251-tyrosine from the peptides formed. Peptide Analysis of the LDL Degradation Products-To study the lZ51-labeledpeptides formed by the granule chymase, fractions 7-11 eluted from the Sephadex G-10 column (Fig. 5B) were pooled and applied to a column of Sephadex (2-25 (theoretical fractionation range from 5000 to 1000). In this

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TABLE I Amino acid analysis of LDL degradation products Granules (30 pg) were preincubated with o-phenanthroline, soybean trypsin inhibitor, orwith no inhibitors as described in the legend to Fig. 3. In control experiments 10 pg of cu-chymotrypsin, 10 pg of carboxypeptidase A (CPA), or a mixture of the two enzymes were preincubated for 3 h in 100 plof buffer A containing 2.5%(v/v) methanol. The degradation assay was conducted in 250 pl of buffer A containing 500 pg of LDL and 30 pg of o-phenanthroline-treated, trypsin inhibitor-treated, or untreated granules. The control experiments were conducted in a similar manner but with the commercial proteolytic enzymes. After incubation at 37 “C for 20 h, the reactions were stopped by adding 10% (w/v) sulfosalicylic acid to reach a final concentration of 2%. The tubes were incubated a t 4 “C for 30 min and centrifuged a t 12,000 X g for 5 min to remove the precipitated material. The supernatantswere removed and analyzed as described under “Experimental Procedures.”

t NaiZ51

lodo-tyrosine



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FIG. 5. Sephadex G-10 analysis of the labeled degradation products of lz6I-LDL. 2 ml of the peak I1 material obtained from the Bio-Gel A-5m analysis (Fig. 4 A , containing 611,000 cpm) was applied to a Sephadex G-10 column (A).Panels B, C, and D show the corresponding analyses of the peak I1 material represented in Fig. 4 B, C , and D (containing 553,000, 160,000, and 132,000 cpm, respectively). The column (1X 30 cm) equilibrated with buffer A was eluted with the same buffer at a flow rate of 5 ml/h at 4 ”C. Fractions of about 370plwere measured for their “’I radioactivity. The void volume of the column (V,) and the elution positions of commercial Na”’I and commercial iodo-L-tyrosine are indicated by arrows.

Phe 61.3 3.7 Leu 52.1 8.9 Tyr 2.0 39.3 Ile 15.6 Val 6.7 Trp 6.7 Ala 4.2 His 4.2 LYs 1.5 Arg Gly Glu Pro Ser Thr ASP a